Production of glycolate from ethylene glycol and related microbial engineering

ABSTRACT

Processes, systems and microorganisms are described herein for producing glycolate from ethylene glycol. The processes generally comprise supplying a fermentation broth into a fermentation vessel, wherein the fermentation broth comprises ethylene glycol and a microorganism having a functional metabolic pathway for utilizing ethylene glycol as a carbon source. In a growth phase, an oxygen-containing gas is injected into the fermentation broth to provide oxygen bio-availability conditions to promote cell growth of the microorganism and limit accumulation of glycolate in the fermentation broth. In a production phase, an oxygen-containing gas is injected into the fermentation broth to provide oxygen bio-availability conditions to promote production of glycolate from ethylene glycol by the microorganism and accumulation of the glycolate in the fermentation broth, to produce a glycolate enriched broth.

TECHNICAL FIELD

The technical field generally relates to the production of glycolatefrom ethylene glycol, particularly using certain microorganisms as wellas cultivation conditions and processing techniques.

BACKGROUND

Glycolic acid is an alpha-hydroxy acid used in the manufacture ofbiodegradable polymers such as polyglycolic acid (PGA),polylactic-glycolic acid (PLGA) and other degradable polymers, as wellas being used as an ingredient in a number of industrial and householdproducts such as solvents, paints, and especially cosmetics. Today,commercial production of glycolic acid is largely produced through theuse of petrochemical feedstocks and by using highly toxic startingmaterials such as formaldehyde. Hence, it is desirous to produce thisimportant chemical from a non-toxic, renewable source.

In contrast, some chemicals can be produced electrochemically. Ethyleneglycol is one such chemical that is also a promising feedstock forbioprocesses because it can be derived from CO₂ and for which a processhas been developed (Tamura et al. 2015). In this regard, its utilizationas a feedstock for biological processes is important because it canserve as a replacement for glucose in modern bioprocesses such as thoseproduced from point source emissions.

Some other bioprocesses also exist for producing glycolic acid. Theseconventional approaches to glycolic acid by genetically modifiedmicroorganisms have instead focused on using sugars as the substrate forproduction. Several studies have been published that have examinedglycolic acid production from glucose and xylose (Deng et al. 2015;Alkim et al. 2016; Koivistoinen et al. 2013; Zahoor et al. 2014; Cam etal. 2016). The highest of these reports achieves titers of 56.44 g/L anda yield of 0.52 g/g. However, the use of sugar feedstocks presentslimitations such as it does not allow the capture of point source carbonemissions.

Thus, a possible advantage of embodiments disclosed herein can be toprovide a biological method for glycolate production that uses a carbonfeedstock that can be derived renewably and does not utilize toxiccompounds such as formaldehyde. Secondly, whereas previously developedbiological methods for producing glycolate have been described in theliterature, large-scale production of glycolate using those methods havecertain drawbacks. For example, the production of glycolic acid fromethylene glycol by biological methods has relied on the use of aco-substrate to provide cell growth or to induce expression of theethylene glycol metabolizing enzymes. The use of co-substrates canpresent certain challenges for large-scale glycolate production over theuse of a single substrate, such as additional cost. Moreover, otherbiological methods for producing glycolic acid have been performed underneutral pH conditions. Whereas the production of glycolic acid lowersthe pH of the fermentation broth, it would be preferable to have aculturing environment less than pH 7.2 such that the costs of glycolicacid production can be decreased as less buffer is required for themedia to maintain a neutral pH.

For example, the biological production of glycolic acid has beendescribed by various authors. However, previous methods have a number ofdrawbacks. Previous knowledge on the conversion of ethylene glycol toglycolic acid by a natural or genetically modified microorganism hasrelied on the oxidation of ethylene glycol in a phosphate bufferedmedium or in distilled water and has relied on a resting cellbiotransformation for the accumulation of glycolic acid. This hascertain disadvantages, such as requiring the separation of biomass fromthe culturing media followed by resuspension of that biomass into freshmedia for resting cell transformation at much larger cell densities.Thus, a disadvantage of such processes is the need for additionalequipment like a secondary vessel to carry out biotransformation oradditional centrifuges for cell separation and concentration.

In addition, several previous methods rely on using ethylene glycol as asecondary carbon source for biotransformation, in addition to a primarycarbon source for growth such as glucose, sorbitol or even propyleneglycol. The reliance on a secondary carbon source for growth can be anadditional cost for the process.

A disadvantage of other previous methods for producing glycolic acidemploying genetically modified microorganisms is that they employ oxygensensitive enzymes. The production of glycolic acid requires oxygen as asubstrate. However, under high oxygen concentrations or mass transferrates such as those that might be expected in an industrial bioreactor,it is necessary that the microorganism remain viable by havingfunctional enzymes. Hence, the use of oxygen sensitive enzymes forproducing glycolic acid can have a detrimental effect on theproductivity and titres of the process.

In several alternative known methods, glycolic acid production occurs ata pH near or above 7. When organic acids are produced during afermentation, the result is a drop in pH of the fermentation broth.Hence, it is more economically viable to operate the fermentation at alower pH since it requires the addition of less base or buffer to thefermentation medium. Therefore, a disadvantage of several alternativesis that they operate at a pH near to or greater than neutral.

Thus, there is a need for technologies that overcome or mitigate atleast some of the disadvantages of known methods.

SUMMARY

Various aspects, implementations, embodiments and features of theinvention are described herein.

In some implementations, the invention relates to the development of amicroorganism and the cultivation conditions for the microorganisms togrow on ethylene glycol and produce glycolic acid. In someimplementations, the invention relates to methods of producing glycolicacid from a substrate substantially comprising ethylene glycol using amicroorganism, which may have been previously genetically engineered tohave certain characteristics, and using certain process operatingconditions.

Described herein are methods for producing glycolic acid, by culturinggenetically modified microorganisms in the presence of ethylene glycolas the sole carbon source for growth and for glycolate production. In apreferred embodiment, air is introduced into the fermentation vesselsuch that the oxygen uptake rate (OUR) is greater than about 6mmol/gDW/h to promote cellular respiration and then a second set ofculturing conditions is established wherein the oxygen uptake rate islowered to below about 6 mmol/gDW/h such that glycolic acid accumulatedin the fermentation medium at a concentration greater than 1 g/L but ina preferred embodiment greater than 10 g/L.

In some embodiments, the culturing media occurs at a pH less than 7.2but in a preferred embodiment where the pH is less than about 6.5(during the production phase).

In some embodiments, the genetically modified microorganism comprises afunctional metabolic pathway for converting ethylene glycol to pyruvate,wherein that metabolic pathway comprises an alcohol dehydrogenase thatis tolerant to oxygen with enhanced activity that converts ethyleneglycol to glycolaldehyde and an aldehyde reductase with enhancedactivity that converts glycolaldehyde to glycolic acid.

In some embodiments, the method for producing glycolic acid fromethylene glycol comprises an active and functional endogenous glycolateoxidase whose activity may be dynamically controlled through the use ofa combination of mechanisms that affect the gene promoter, geneinactivation by protein degradation and/or gene inactivation byallosteric control.

In some aspects of the method for the production glycolic acid, theconcentration of genetically modified microorganisms in the fermenter indry mass is less than about 10 g/L.

In some embodiments, the glycolic acid obtained during the productionphase is greater than 50% yield by mass on ethylene glycol butpreferably greater than 80%.

In some embodiments of the method for producing glycolic acid, thefermentation can be separated into two distinct phases dominated by aprimary growth phase where there is little glycolic acid production anda second phase dominated glycolic acid production and there is littlebiomass production.

In some implementations, the present invention allows for the productionof glycolic acid without the use of a secondary carbon source such asglucose since ethylene glycol serves as both a growth substrate as wellas the precursor for producing glycolic acid. This has significantcommercial benefits because it allows the fermentation to occur in asingle vessel, without the need to separate the genetically modifiedmicroorganisms from its growth media. This simplification allowsproduction of glycolic acid to require fewer fermentation andbiotransformation vessels which would reduce the capital costs of theprocess.

In some implementations, the present invention utilizes an oxygentolerant version of an alcohol dehydrogenase to catalyze the first stepof the metabolic pathway for converting ethylene glycol to glycolicacid.

In some implementations, the present invention employs a two-stagefermentation method wherein genetically modified microorganisms arecultured at a neutral pH but wherein the glycolate production phaseoccurs at a pH less than 7, preferably about 6.5.

In some implementations, the present invention includes a method forproducing glycolic acid by culturing a genetically modified organismsuch as E. coli cells in the presence of ethylene glycol.

In another implementation, there is provided a process for producingglycolate from ethylene glycol, comprising: supplying a fermentationbroth into a fermentation vessel, wherein the fermentation brothcomprises ethylene glycol and a microorganism genetically engineered forincreased conversion of ethylene glycol to glycolaldehyde (andeventually glycolate) in the presence of oxygen as compared to acorresponding microorganism lacking the genetic engineering, thegenetically engineered microorganism being responsive to a decrease inoxygen bio-availability to transition from a cell growth promotingmetabolic pathway in which conversion of glycolate to glyoxylate ispromoted to a glycolate production metabolic pathway in which theconversion of glycolate to glyoxylate is inhibited; in a growth phase,injecting an oxygen-containing gas into the fermentation broth andproviding initial oxygen bio-availability conditions to utilize the cellgrowth promoting metabolic pathway to promote cell growth of themicroorganism and limit accumulation of glycolate in the fermentationbroth; in a production phase, injecting an oxygen-containing gas intothe fermentation broth and providing reduced oxygen bio-availabilityconditions to utilize the glycolate production metabolic pathway topromote production of glycolate from ethylene glycol by themicroorganism and accumulation of the glycolate in the fermentationbroth, to produce a glycolate enriched broth; and recovering at least aportion of the glycolate from the glycolate enriched broth.

In some implementations, there is provided a process for producingglycolate, comprising: supplying a fermentation broth into afermentation vessel, wherein the fermentation broth comprises ethyleneglycol and a microorganism having a functional metabolic pathway forutilizing ethylene glycol as a carbon source; in a growth phase,injecting an oxygen-containing gas into the fermentation broth andproviding oxygen bio-availability conditions to promote cell growth ofthe microorganism and limit accumulation of glycolate in thefermentation broth; and in a production phase, injecting anoxygen-containing gas into the fermentation broth and providing oxygenbio-availability conditions to promote production of glycolate fromethylene glycol by the microorganism and accumulation of the glycolatein the fermentation broth, to produce a glycolate enriched broth.

In some implementations, the microorganism has a functional metabolicpathway for converting ethylene glycol to pyruvate. The functionalmetabolic pathway can include polypeptides catalyzing reactions: (a)ethylene glycol to glycolaldehyde; (b) glycolaldehyde to glycolate; and(c) glycolate to glyoxylate. One or more of the polypeptides can beencoded by one or more polynucleotides that are exogenous and/orheterologous with respect to the microorganism. Expression of one ormore of the polynucleotides can be under control of one or moreregulatory elements. One or more of the regulatory elements can enablecontrol of expression of one or more of the polynucleotides in responseto oxygen levels, pH, nutrient concentrations such as phosphate ornitrogen, the presence or concentration of an inducer, and/or anotherparameter controllable during fermentation. One or more of theregulatory elements can include one or more promoters and/or terminatorsoperably linked to the one or more polynucleotides. One or more of theregulatory elements can be exogenous and/or heterologous with respect tothe microorganism. One or more of the polynucleotides can be comprisedin a plasmid. One or more of the polynucleotides can be integrated intothe genome of the microorganism.

In some implementations, the polypeptide catalyzing reaction (a)comprises an enzyme of class E.C. 1.1.1,

E.C. 1.1.3, or E.C. 1.1.5, or a functional variant or fragment thereof,which converts ethylene glycol to glycolaldehyde. The functional variantcan be a variant having reduced sensitivity to oxygen. The reducedsensitivity to oxygen can include reduced sensitivity to metal catalyzedoxidation. The polypeptide catalyzing reaction (a) can includelactaldehyde reductase. The lactaldehyde reductase can be encoded by thegene fucO. The lactaldehyde reductase can include an amino acidsubstitution I7L and/or L8V or LBM, based on the amino acid numbering ofthe native lactaldehyde reductase encoded by fucO from E. coli

In some implementations, the polypeptide catalyzing reaction (a)comprises an enzyme that uses an oxygen-insensitive cofactor. The enzymecan use a cofactor other than iron, which can be zinc (e.g., azinc-dependent alcohol dehydrogenase or an NAD-dependent alcoholdehydrogenase).

In some implementations, the polypeptide catalyzing reaction (b)comprises an enzyme of class E.C. 1.2.1, E.C. 1.2.3, or E.C. 1.2.5, or afunctional variant or fragment thereof which converts glycolaldehyde toglycolate. The polypeptide catalyzing reaction (b) can includelactaldehyde dehydrogenase. The lactaldehyde dehydrogenase can beencoded by the gene aldA.

In some implementations, the polypeptide catalyzing reaction (c) caninclude an enzyme of class E.C. 1.1.3.15, or a functional variant orfragment thereof, which converts glycolate to glyoxylate. In someimplementations, the polypeptide catalyzing reaction (c) comprisesglycolate oxidase.

In some implementations, the microorganism further comprises apolynucleotide encoding a polypeptide catalyzing reaction (d) export ofintracellular glycolate to the extracellular environment. Thepolypeptide catalyzing reaction (d) can be exogenous and/or heterologouswith respect to the microorganism.

In some implementations, the microorganism comprises a polynucleotideencoding a polypeptide that catalyzes both reactions (a) and (b).

In some implementations, the microorganism further comprises one orpolynucleotides encoding one or more enzymes for converting glycolate topolyglycolic acid, ethanolamine, or glycine. The microorganism furthercan include an exporter of polyglycolic acid, ethanolamine, or glycinethat exports intracellular polyglycolic acid, ethanolamine, or glycineto the extracellular environment.

In some implementations, the microorganism is bacteria (e.g.,Escherichia coli) which can be genetically modified for improvedtolerance to acidic pH, as compared to corresponding wild-type bacteria.

In some implementations, the microorganism is a yeast or fungus; or ayeast or a fungus that is genetically modified for improved tolerance toacidic pH, as compared to a corresponding wild-type yeast or fungus. Theyeast or fungus can be from the species Candida boidinii, Candidaetchellsii, Candida geochares, Candida lambica, Candida sorbophila,Candida sorbosivorans, Candida sorboxylosa, Candida vanderwaltii,Candida zemplinina, Debaryomyces castellii, Issatchenkia orientalis,Kluyveromyces lactis, Kluyveromyces marxianus, Pichia anomala, Pichiajadinii, Pichia jadinii, Pichia membranifaciens, Saccharomyces bayanus,Saccharomyces bulderi, Saccharomycopsis crataegensis, Zygosaccharomycesbisporus, Zygosaccharomyces kombuchaensis, or Zygosaccharomyces lentus.

In some implementations, the microorganism is from Pseudomonas species,Clostridium species, Chlorella species or other algae, Gluconobacteroxydans, Pichia naganishii, Corynebacterium species, or Corynebacteriumglutamicum; or from a microorganism thereof that is genetically modifiedfor improved tolerance to acidic pH, as compared to a correspondingwild-type microorganism.

In some implementations, the microorganism is from Haloferaxmediterranei, Halobactreium salinarum, Nicotiana tabacum, or Thermusthermophilus; or from a microorganism thereof that is geneticallymodified for improved tolerance to acidic pH, as compared to acorresponding wild-type microorganism.

The microorganism can be for use in the fermentative production ofglycolate, polyglycolic acid, ethanolamine, and/or glycine. In someimplementations, there is provided a use of the microorganism as definedabove or herein for the fermentative production of glycolate,polyglycolic acid, ethanolamine, and/or glycine.

In some implementations of processes described herein, the main carbonsource for the microorganism is the ethylene glycol. In addition, theonly carbon source for the microorganism can be the ethylene glycol.

In some implementations of processes described herein, the microorganismis genetically engineered for increased conversion of ethylene glycol toglycolaldehyde (and eventually glycolate) in the presence of oxygen ascompared to a corresponding microorganism lacking the geneticengineering, and is responsive to a decrease in oxygen bio-availabilityto transition from a cell growth promoting metabolic pathway in whichconversion of glycolate to glyoxylate is promoted to a glycolateproduction metabolic pathway in which the conversion of glycolate toglyoxylate is inhibited.

In some implementations, the process includes recovering at least aportion of the glycolate from the glycolate enriched broth. Therecovering can include removing the glycolate enriched broth from thefermentation vessel, separating cells from the glycolate enriched brothto produce a biomass-depleted broth, and producing a glycolate enrichedstream from the biomass-depleted broth.

In some implementations, the process includes recycling or reusing atleast a portion of the cells separated from the glycolate enriched brothback into the fermentation vessel. In some implementations, the processincludes releasing a liquid portion of the glycolate enriched broth fromthe fermentation vessel, and producing a glycolate enriched stream fromthe liquid portion. In some implementations, the process includesretaining cells within the fermentation vessel, replenishing thefermentation vessel with additional broth and ethylene glycol, andreusing the retained cells for additional production of glycolate.

In some implementations, the glycolate production is conducted as abatch or fed-batch process.

In some implementations, the fermenter vessel has a chamber in which thefermentation broth is located and in which the microorganism cell growthand glycolate production both occur. In some implementations, there isno additional step to carry out biotransformation after fermentation.The oxygen-containing gas can include or be air. In someimplementations, the oxygen-containing gas is introduced during thegrowth phase at a sufficiently elevated concentration to inhibitextracellular accumulation of glycolate in the fermentation broth. Theoxygen-containing gas can be introduced during the production phase at asufficiently low concentration to inhibit metabolic conversion ofglycolate into a corresponding metabolite. The oxygen-containing gas canbe introduced during the growth phase above an oxygen bio-availabilityminimum threshold, and during the production phase below an oxygenbio-availability maximum threshold.

In some implementations, the oxygen bio-availability minimum thresholdand the oxygen bio-availability maximum threshold are predetermined. Theoxygen bio-availability minimum threshold and the oxygenbio-availability maximum threshold can be the same value. The oxygenbio-availability minimum threshold can be between 4 mmol/gDW/h and 8mmol/gDW/h. The oxygen bio-availability maximum threshold can be between4 mmol/gDW/h and 8 mmol/gDW/h and can be below the oxygenbio-availability minimum threshold. The oxygen bio-availability maximumthreshold can be greater than about 6 mmol/gDW/h, and the oxygenbio-availability minimum threshold can be below about 6 mmol/gDW/h;and/or the oxygen bio-availability maximum and minimum thresholds can bedifferent from each other by at least 0.5 mmol/gDW/h, by at least 1mmol/gDW/h, by at least 2 mmol/gDW/h, by at least 3 mmol/gDW/h, by atleast 4 mmol/gDW/h, and/or by at most 4 mmol/gDW/h.

In some implementations, the production phase is operated and controlledsuch that a concentration of extracellular glycolate in the fermentationbroth is greater than about 1 g/L, optionally greater than 2 g/L, 5 g/L,7 g/L, 10 g/L or 15 g/L, prior to removal of the glycolate from thefermentation broth. In some implementations, the growth phase isconducted at a pH of about 7, optionally at a pH of about 6.5 to about7.2, still further optionally at a pH of less than 7.2.

In some implementations, the production phase is conducted at a pH ofless than 7, optionally at a pH of less than 6.5, and still furtheroptionally at a pH of about 6 to about 6.9 . In some implementations,the growth phase is conducted at a temperature greater than 30° C.,optionally between 30° C. and 42° C., between 33° C. and 40° C., between36° C. and 38° C., or at about 37° C.

In some implementations, the production phase is conducted at atemperature greater than 30° C., optionally between 30° C. and 42° C.,between 33° C. and 40° C., between 36° C. and 38° C., or at about 37°C.; or still further optionally at a temperature that is generally thesame as that of the growth phase. In some implementations, the growthphase is conducted at an air injection rate that is determined based ondesign of the fermentation vessel and the desired oxygenbio-availability minimum threshold.

In some implementations, the production phase is conducted at an airinjection rate that is determined based on design of the fermentationvessel and the desired oxygen bio-availability maximum threshold.

In some implementations, the growth phase is conducted at a pH of about7 and/or a temperature of about 37° C. The production phase can beconducted at a pH of about 6.5 and/or at a pH that is about 0.5 lowerthan that of the growth phase, and/or a temperature of about 37° C.and/or a temperature generally the same as that of the growth phase.

In another implementation, there is provided a process for producingglycolate, comprising: providing a fermentation broth comprising acarbon source and a microorganism, wherein the ethylene glycol is aprimary component of the carbon source; and providing fermentationconditions to induce conversion the ethylene glycol into glycolate bythe microorganism, and accumulation of the glycolate in the fermentationbroth. In such an implementation, the process can include one or morefeatures from any one of the previous paragraphs or items describedherein.

In another implementation, there is provided a process for producingglycolate by microbial conversion of ethylene glycol into glycolateusing a microorganism that is genetically engineered to consume ethyleneglycol and comprises a polynucleotide encoding a lactaldehyde reductaseand/or a polynucleotide encoding a lactaldehyde dehydrogenase.

In another implementation, there is provided a process for producingglycolate by microbial conversion of ethylene glycol into glycolateusing a microorganism that is genetically engineered for increasedconversion of ethylene glycol into a first corresponding metabolite inthe presence of oxygen as compared to a corresponding microorganismlacking the genetic engineering, and for oxygen-dependent conversion ofglycolate into a second corresponding metabolite.

In such process implementations, the process can include one or morefeatures from any one of the previous paragraphs or items describedherein.

Further background and details regarding optional embodiments, aspects,experiments and examples related to the invention are described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C are diagrams showing metabolic conversion pathwaysof ethylene glycol (FIG. 1A), xylose (FIG. 1B), and glucose (FIG. 1C).

FIG. 2 is a graph of cell growth and substrate consumption over time,particularly MEG concentration and OD₆₀₀ versus fermentation time.

FIGS. 3A and 3B is a pair of graphs showing the influence of aeration onglycolate production, particularly metabolite and cDW concentration aswell as MEG versus time.

FIG. 4 is a graph showing metabolic modelling of glycolate production,in particular glycolate yield (glycolate, solid), respiratory quotient(RQ, dotted) and substrate specific productivity (SSP, dashed) versusoxygen uptake rate, modelled using flux balance analysis (FBA).

FIGS. 5A and 5B are a pair of graphs that relate to fermentationprofiles for fed batch strategies, particularly showing glycolate,biomass and ethylene glycol concentrations versus time.

FIGS. 6A and 6B are diagrams showing flux distribution of the metabolismand enzymes in the pathway under aerobic (FIG. 6A) and oxygen limited(FIG. 6B) conditions.

FIG. 7 is a block flow diagram for the production of glycolate fromethylene glycol.

FIG. 8 is a schematic including a block flow diagram, an illustrativegraph of two stage process control, and an illustration of the metabolicpathway for conversion of ethylene glycol to glycolate.

FIG. 9 is a graph of cell growth over time contrasting a wild-typeEscherichia coli MG1655 strain to a corresponding strain expressing theethylene glycol oxidizing Gox0313 enzyme that uses zinc as a cofactorinstead of iron, as well as the glycolaldehyde oxidizing enzyme, aldA,when cultured in M9 minimal media supplemented with 20 g/L ethyleneglycol and 0.1% yeast extract.

DETAILED DESCRIPTION

Ethylene glycol can be used as a substrate for microbial conversion intoglycolate, where the production process can leverage certain features ofthe microorganism and process operating conditions to enhanceperformance.

For instance, the process can be controlled such that oxygenavailability and uptake by the microorganism are varied over time tofacilitate a two-phase process, the first phase being a growth phasepromoting cell growth and limiting glycolate accumulation and the secondphase being a glycolate production phase promoting glycolateaccumulation in a fermentation broth. This two-phase process can befacilitated by controlling oxygen uptake conditions in the fermentationbroth, e.g., by varying air injection rates, such that the growth phasehas high oxygen uptake rates and the production phase has lower oxygenuptake rates.

In addition, the microorganism that is deployed in the process can be agenetically engineered microorganism that has features enabling improvedperformance for glycolate production from ethylene glycol. For instance,the microorganism can be engineered to comprise a functional metabolicpathway for converting ethylene glycol to pyruvate, wherein themetabolic pathway comprises the conversion of ethylene glycol toglycolaldehyde, and the conversion of glycolaldehyde to glycolate.Various microorganisms, such as bacteria or yeast, can be engineeredpursuant to the methods described herein to provide an advantageousmicroorganism for glycolate production.

Furthermore, the process can include ethylene glycol as essentially theonly carbon substrate for microbial growth and glycolate production. Insuch cases, trace amounts of other carbon sources can be present, butethylene glycol is the dominant carbon source with no substantialco-substrates, such as glucose and/or xylose. For instance, the ethyleneglycol can be at least 80 wt %, 85 wt %, 90 wt %, 95 wt %, or 99 wt % or100 wt % of the carbon source for cell growth and/or for glycolateproduction, for example during the growth phase and the productionphase.

Turning to FIG. 7 , a general illustration of an example process isshown. Ethylene glycol and the microorganism are used in thefermentation step, along with an oxygen-containing fluid, such as anoxygen-containing gas like air. The fermentation can be a batch process,for example. At the end of the fermentation reaction, which can includethe two-phase process control mentioned above, a glycolate enrichedsolution is produced and can be subjected to separation to produceglycolate (e.g., at higher concentrations or relatively pure) as well asa spent solution.

In terms of separating the glycolate from the fermentation broth,various methods can be used. For example, separation methods based onreactive extraction, crystallization, adsorption-elution, and so on,could be used. In some implementations, the fermentation broth includingthe biomass is removed from the fermentation vessel and is thensubjected to a series of separation steps, e.g., biomass removalfollowed by glycolate extraction. Alternatively, a portion of thefermentation broth could be removed from the fermentation vessel whilethe biomass remains in the vessel, and then the biomass-depletedfermentation broth can be subjected to a separation technique to removethe glycolate. The unit operations can be designed and implementeddepending on the process being batch, semi-batch or continuous.

It is also noted that the cells can be reused in a continuous process ora batch process. The cells could be separated from the glycolate richfermentation broth, for example by centrifugation or other solid-liquidseparation methods, and then reused in the fermentation vessel. In sucha scenario, the process using recycled cells can be adapted such thatthe two-phase process may not be required as the cells may be ready forreuse in the production phase. Eventually the used cells can be removedfrom the process, for example after their viability period, after whicha new cell culture can be introduced in the fermentation vessel and thetwo-phase process can be repeated. Thus, in some implementations, cellscan be reused in the production phase through multiple batches,depending on viability and stability factors.

Referring to FIG. 8 , a glycolate production system 10 can include afermenter 12 to which a feed line 14 is coupled for feeding the ethyleneglycol. A microorganism inlet 16 can also be provided to supply themicroorganism to the fermenter 12. The fermenter also has side walls 16,a bottom 18 and a top 20 defining a fermentation chamber 22 in which thefermentation reactions occur. The feed materials form a fermentationbroth within the fermentation chamber 22. An oxygen-containing fluidinlet 24 is also coupled to the fermenter 12 for feeding anoxygen-containing fluid into the fermentation broth. Theoxygen-containing fluid can be air, for example, or another gas thatincludes oxygen. Multiple inlets for the various feed materials can alsobe provided. The oxygen-containing fluid inlet 24 can include a gas feedline 26 with a flow control device 28, such as a valve, to enable theflow rate of the gas to be controlled or adjusted. The oxygen-containingfluid inlet 24 can also include a sparging unit that has multiple outletapertures distributed over the cross-section of the fermentation chamber22 to inject gas bubbles into the fermentation broth.

FIG. 8 also shows that the system 10 can include various measurementdevices coupled to the fermenter 12 to measure certain variables, suchas temperature (T), pH, concentration (C), and so on. The system 10 canalso include a controller 32 that can be coupled to various measurementdevices to receive information, and also to control units of thefermenter 12 in order to modify one or more process operatingconditions. For instance, the controller 32 can be configured to receiveinformation regarding the cell growth progression of the microorganismin the fermentation broth, and to reduce the injection rate of the airby closing the valve 28 once the cell growth has sufficientlyprogressed, thereby initiating the second phase of the process. Thecontroller 32 can also be configured to regulate the temperature byvarying heat that is provided to the system, to regulate the pH byaddition of a pH modifier to keep the pH within a desired window, and/orto initiate different phases of the process including the growth phase,the production phase, and then the recovery phase once a desiredconcentration of glycolate has been produced.

Various methods and variables can be used to monitor cell growthprogression. For example, CO₂ off gas could be measured to determinegrowth progression. The monitoring is preferably based on measuring cellquantity, although other variables could be measured. Characteristics ofthe fermentation broth could be measured to infer cell growthprogression. In addition, cell growth progression could be determinedusing various other techniques, or could be estimated based on previousexperiments or operations such that active determination is notconducted but is rather estimated based on variables such asfermentation time.

Furthermore, in terms of monitoring or determining the impact of oxygenon the system, methods can be used to assess the bio-availability ofoxygen and can include the amount of oxygen in the fermentation vesselas well as the mass transfer of the oxygen from the gas phase to theliquid phase. In some embodiments, another gas (e.g., carbon dioxide)may be measured as a proxy for oxygen levels. Bio-availability of oxygencan be controlled by adjusting gas feed rate into the vessel, rate ofmixing in the vessel, temperature in the vessel or other parameters thatimpact solubility and dissolved oxygen, and/or oxygen content in the gasfed into the vessel (e.g., by co-feeding pure oxygen and/or purenitrogen with air via the same inlet or separate inlets to increase ordecrease oxygen content in the feed gas), and so on. Oxygenbio-availability can be estimated and controlled within each processphase, and also in order to transition from one phase to the other.

Other operating parameters can also be monitored and manipulated duringthe process. For example, pH can be changed when transitioning fromgrowth to production phase. In addition, agitation could be changedduring this transition, as well as temperature. Such parameters can bechanged in order to impact the bio-availability of oxygen and/or to haveother beneficial impacts on the process phase of interest.

FIG. 8 illustrated a close-up view of some of the chemical reactionsthat occur, notably the metabolic conversion of ethylene glycol intoglycolaldehyde via alcohol dehydrogenase, followed by the metabolicconversion of glycolaldehyde into glycolate via aldehyde reductase. Moreregarding certain aspects and features of the microorganisms that can beused in the process is described further below.

Referring still to FIG. 8 , once the fermentation is complete, theglycolate enriched solution can be evacuated from the fermenter 12 viaan outlet line 34 and supplied to a separator 36 or other downstreamprocessing units to produce a glycol stream 38 and a spent solution 40.Various downstream separation units can be used to separate theglycolate, e.g., filtration or centrifugation to remove biomass/cells,and then reactive extraction, crystallization, chromatography, and soon, to remove glycolate.

Microorganisms

The production of glycolate from ethylene glycol can be facilitated bythe use of a genetically engineered microorganism having certaincharacteristics. For example, a microorganism can be engineered tocomprise a functional metabolic pathway for converting ethylene glycolto pyruvate, wherein the metabolic pathway comprises polypeptidescatalyzing (a) the conversion of ethylene glycol to glycolaldehyde, (b)the conversion of glycolaldehyde to glycolate, and (c) the conversion ofglycolate to glyoxylate. The metabolic pathway may further comprisenative and/or exogenous enzymes enabling the conversion of glyoxylate topyruvate.

In some implementations, the microorganism has the ability to utilizeethylene glycol as a sole or dominant carbon source. By dominant carbonsource, it is meant that the microorganism utilizes ethylene glycolprimarily over other carbon sources such as glucose and/or xylose tosupport growth and/or chemical production (e.g., glycolate, ordownstream glycolate metabolization products). As used herein, theexpressions “microorganism has the ability to utilize ethylene glycol asa sole carbon source”, “microorganism can utilize ethylene glycol as asole carbon source”, and “microorganism uses ethylene glycol as a solecarbon source” are used interchangeably as referring to a property orcharacteristic of the microorganism itself, and not necessarily to thecontent of the fermentation broth in which the microorganism is beingemployed.

In some implementations, the microorganism described herein isgenetically engineered to exhibit sustained growth on ethylene glycol asthe sole or main carbon source. In some implementations, the geneticallyengineered microorganism may exhibit at least 2-fold, 3-fold, 4-fold,5-fold, 6-fold or more growth over a corresponding wild-type (ornon-genetically engineered) microorganism, for example as measured at 24hours post-inoculation by optical density (O.D.) at 600 nm, and/or in agrowth phase of a process described herein. In some implementations, thegenetically engineered microorganism has the ability to increase itscell density (number of cells per unit volume) by a factor of 2, 3, 4,5, or 6, as compared to a corresponding wild-type (or non-geneticallyengineered) microorganism, when cultured in the presence of ethyleneglycol as the sole or main carbon source, such as after initialnon-ethylene glycol carbon sources are depleted from thestarting/inoculation culture medium, when cultured in a growth phase ofa process described herein.

In some implementations, the microorganism may be bacteria (e.g.,Escherichia coli), that may be further genetically modified for improvedtolerance to acidic pH, as compared to corresponding wild-type bacteria.

In some implementations, the microorganism may be a yeast or fungus,such as from the species Candida boidinii, Candida etchellsii, Candidageochares, Candida lambica, Candida sorbophila, Candida sorbosivorans,Candida sorboxylosa, Candida vanderwaltii, Candida zemplinina,Debaryomyces castellii, Issatchenkia orientalis, Kluyveromyces lactis,Kluyveromyces marxianus, Pichia anomala, Pichia jadinii, Pichia jadinii,Pichia membranifaciens, Saccharomyces bayanus, Saccharomyces bulderi,Saccharomycopsis crataegensis, Zygosaccharomyces bisporus,Zygosaccharomyces kombuchaensis, or Zygosaccharomyces lentus. Suchspecies have been shown to exhibit improved tolerance to low pHenvironments. In some implementations, the yeast or fungus may befurther genetically modified for improved tolerance to acidic pH, ascompared to corresponding wild-type bacteria.

In some implementations, the microorganism may be from a species thatnaturally consumes ethylene glycol. Such species include for examplePseudomonas species (e.g., Pseudomonas putida; Muckschel et al., 2012),Clostridium species (Gaston and Stadtman, 1963), Chlorella species orother algae (Kishi et al., 2015), Gluconobacter oxydans (Zhang et al.,2016), or Pichia naganishii (Kataoka et al., 2001). In someimplementations, the microorganism from such a species may be furthergenetically modified for improved tolerance to acidic pH, as compared tocorresponding wild-type bacteria.

In some implementations, for example wherein the microorganism is from aspecies that naturally consumes ethylene glycol, the microorganism maybe genetically modified to disrupt endogenous metabolic pathways forethylene glycol uptake and/or utilization. Alternatively, themicroorganism's native metabolic pathways for ethylene glycol uptakeand/or utilization may be diverted to produce glycolate. Microorganismsthat natively consume ethylene glycol may utilize one of two types ofmetabolic pathways.

The first pathway of ethylene glycol uptake/degradation utilizes adiol-dehydratase resulting in the dehydration of ethylene glycol toacetaldehyde. Acetaldehyde is then activated to acetyl-CoA by anacetaldehyde dehydrogenase enzyme which provides the cell with the keyprecursor metabolite to support growth via the TCA cycle andgluconeogenic pathways. The production of one mole of acetyl-CoA fromone mole of ethylene glycol concomitantly produces one NADH. Thispathway is most commonly found in some Clostridium species and a fewother anaerobic organisms owing to the oxygen sensitivity of thediol-dehydratase^(25,27). Accordingly, in some implementations, themicroorganism may comprise one or more (exogenous) polynucleotidesencoding enzymes (e.g., a diol-dehydratase) that converts ethyleneglycol to acetaldehyde. The microorganism may comprise one or more(exogenous) polynucleotides encoding an enzyme (e.g., an acetaldehydedehydrogenase) that converts acetaldehyde to acetyl-CoA.

The second pathway of ethylene glycol uptake/degradation utilizes apathway wherein ethylene glycol is successively oxidized usingnicotinamide cofactors and oxygen to produce glyoxylate. Glyoxylatewhich is a gluconeogenic carbon substrate, can then be used as thegrowth metabolite as it enters lower glycolysis at the2-phosphoglycerate node as well as the TCA cycle via the glyoxylateshunt. Accordingly, in some implementations, the microorganism can begenetically modified to express one or more of (exogenous) enzymes ofthis second pathway. In some implementations, the microorganism may begenetically modified to express one or more (exogenous) polynucleotidesencoding one or more enzymes that convert glyoxylate to glycolate (e.g.,glyoxylate reductase; EC 1.1.1.26); and/or engineer the microorganism todisrupt or delete a native glycolate oxidase enzyme.

In some implementations, the microorganism may be from a Corynebacteriumspecies (e.g. Corynebacterium glutamicum).

In some implementations, the microorganism may be from Haloferaxmediterranei, Halobactreium salinarum, Nicotiana tabacum, or Thermusthermophilus. Such organisms have the ability to grow under extremeconditions, which may be advantageous under certain implementations.

Genetic Modifications

In some implementations, the one or more polypeptides catalyzingreactions (a), (b) or (c) may be encoded by one or more polynucleotidesthat are either endogenous or exogenous and/or heterologous with respectto the microorganism.

As used herein, “endogenous” with respect to genetic components of themicroorganism means the genetic component (polynucleotide, regulatoryelement, promoter, or terminator) is present at a particular location inthe genome of a native form of a particular organism. In contrast,“exogenous” as used herein with regard to genetic components means thatthe genetic component is not present at a particular location in thegenome of a native (i.e., wild-type or naturally-occurring) form of aparticular organism. For example, an exogenous genetic component mayhave either a native or a non-native polynucleotide sequence. A geneticcomponent having a native polynucleotide sequence (i.e., a sequence thatis present in the genome of the corresponding wild-type organism), wouldstill be considered as an exogenous genetic component if it were presentat a different location in the genetically engineered microorganism thanthe location of the same genetic component within the genome of thewild-type microorganism. For further clarity, a polynucleotide sequencethat is native to a first microorganism species would be considered asexogenous if it were introduced into the genome of a secondmicroorganism species, and vice versa.

As used herein, the term “heterologous” with respect to polynucleotidesand polypeptides means that the sequences of the polynucleotides andpolypeptides are not normally found in the corresponding native orwild-type microorganism that is being genetically modified.

In some implementations, the expression of one or more of thepolynucleotides described herein may be placed under control of one ormore regulatory elements (e.g., promoters, terminators, transcriptionalenhancers, activators, or repressors, genetic switches). In someimplementations, such regulatory elements (which may be exogenous and/orheterologous with respect to the microorganism) are operably linked to apolynucleotide described herein to enable control of its expression inresponse to changes in oxygen levels (e.g., an oxygen-sensitivepromoter), intracellular or extracellular pH (e.g., a pH-sensitivepromoter), nutrient concentrations (e.g., phosphate or nitrogen), thepresence or concentration of an inducer (e.g., an inducible promoter),and/or a parameter controllable during fermentation (e.g., temperature,composition of the fermentation broth). In some implementations, acombination of different regulatory elements may be used to achievedifferential expression of one or more polynucleotides described herein,for example, depending on the phase of the processes described herein(e.g., growth phase versus production phase). For example, expression ofa polypeptide catalyzing the conversion of glycolate to glyoxylate canbe increased during a growth phase, and subsequently decreased during aproduction phase, while expression of a polypeptide encoding a glycolateexporter may be decreased during the growth phase and subsequentlyincreased during the production phase.

In some implementations, one or more of the polynucleotides describedherein may by comprised in a plasmid, and/or integrated into the genomeof the microorganism. Where the polynucleotides are comprised in aplasmid, the plasmid may further comprise one or more selection markers(e.g., antibiotic resistance genes) that enable selection and/oridentification of positive transformants.

(a) Conversion of Ethylene Glycol to Glycolaldehyde

In some implementations, the microorganism may be genetically engineered(or genetically modified) for increased conversion of ethylene glycol toglycolaldehyde in the presence of oxygen (e.g., improved oxygentolerance) as compared to a corresponding microorganism lacking thegenetic engineering (or genetic modification). For example, themicroorganism may be genetically engineered (or genetically modified)for improved oxygen-tolerant conversion of ethylene glycol toglycolaldehyde as compared to a corresponding microorganism lacking thegenetic engineering (or genetic modification). As used herein, theexpression “improved oxygen tolerant conversion” or “improved oxygentolerance” relates a genetic modification that alleviates the negativeeffect of increased/increasing oxygen levels on the uptake and/orconsumption of ethylene glycol by microorganism that, for example,relies on enzymes susceptible to degradation by metal-catalyzedoxidation.

In some implementations, the microorganisms described herein maycomprise a polynucleotide encoding a polypeptide that catalyzes reaction(a), i.e., the conversion of ethylene glycol to glycolaldehyde, whereinthe polypeptide comprises an enzyme of class E.C. 1.1.1, E.C. 1.1.3, orE.C. 1.1.5, or a functional variant or fragment thereof.

Enzymes belonging to E.C. 1.1.1 include oxidoreductases acting on theCH-OH group of donors with NAD(+) or NADP(+) as acceptor (Levin et al.,2004). Enzymes belonging to E.C. 1.1.3 include oxidoreductases acting onthe CH-OH group of donors with oxygen as acceptor (e.g., oxidases suchas alcohol oxidase or glycerol oxidase; Isobe, 1995; Isobe and Nishiseb,1995). Enzymes belonging to E.C. 1.1.5 include oxidoreductases acting onthe CH—OH group of donors with a quinone or similar compound as acceptor(e.g., a pyrroloquinoline quinone (PQQ)-dependent enzyme, such as fromPseudomonas putida; Muckschel et al., 2012).

As used herein, the expressions “functional variant” and “functionalfragment thereof” refer to variants of polypeptides described hereinthat differ from a reference polypeptide by one or more amino acidsubstitutions, deletions, and/or insertions that do not abrogate thedesired enzymatic activity of the polypeptide. For example, in someimplementations, a functional variant may comprise one or moreconservative amino acid substitutions of a reference polypeptide, or afunctional fragment may comprise a truncation at the N and/or C terminusof a reference polypeptide without affecting for example a catalyticdomain of the reference polypeptide.

In some implementations, a functional variant of a polypeptide describedherein may have an improved property (e.g., improved enzymatic activity,stability and/or subcellular localization) over the referencepolypeptide, for the purposes of the processes described herein (e.g.,for enhanced glycolate production). For example, the functional variant(e.g., of a polypeptide catalyzing reactions (a) and/or (b)) may havereduced sensitivity (improved tolerance) to oxygen (e.g., to metalcatalyzed oxidation).

In some implementations, the expression and/or activity of the enzymethat converts ethylene glycol to glycolaldehyde is oxygen-independent orhas reduced oxygen-sensitivity (improved oxygen tolerance), for exampleas compared to a corresponding wild-type enzyme.

In some implementations, the polypeptide that catalyzes reaction (a) maycomprise or consist of lactaldehyde reductase, also known as propanedioloxidoreductase E.C. 1.1.177, or a functional variant thereof In someimplementations, the lactaldehyde reductase may encoded by the genefucO. In some implementations, the lactaldehyde reductase may comprisean amino acid substitution I7L and/or L8V or LBM, based on the aminoacid numbering of the native lactaldehyde reductase encoded by fucO fromE. coli MG1655. Such amino acid substitutions may improve the resistanceof the enzyme to degradation by metal catalyzed oxidation, therebyreducing its sensitivity to oxygen.

In some implementations, the polypeptide that catalyzes reaction (a) maycomprise or consist of an enzyme that uses an oxygen-insensitive oroxygen less sensitive cofactor, such as a cofactor other than iron. Insome implementations, the polypeptide that catalyzes reaction (a) maycomprise or consist of an enzyme that uses zinc as a cofactor and/or theenzyme is or comprises as a zinc-dependent alcohol dehydrogenase (e.g.,a cinnamyl alcohol dehydrogenase), such as the NAD-dependent alcoholdehydrogenase from Gluconobacter oxydans 621H set forth in Genbankaccession: AAW60096, as shown in FIG. 9 .

In some implementations, the expression of the polynucleotide encodingthe polypeptide that catalyzes reaction (a) may be placed under thecontrol of a constitutively active promoter.

In some implementations, expression of the polypeptide catalyzingreaction (b) may be deliberately controlled or attenuated to maintainthe intracellular levels of glycolaldehyde to sub-toxic levels (e.g., bythe use of a weak promoter, an inducible promoter, and/or low-copynumber plasmid).

(b) Conversion of Glycolaldehyde to Glycolate

In some implementations, the microorganisms described herein maycomprise a polynucleotide encoding a polypeptide that catalyzes reaction(b), i.e., the conversion of glycolaldehyde to glycolate, wherein thepolypeptide comprises an enzyme of class E.C. 1.2.1, E.C. 1.2.3, or E.C.1.2.5, or a functional variant or fragment thereof.

Enzymes belonging to E.C. 1.2.1 include oxidoreductases acting on thealdehyde or oxo group of donors with NAD(+) or NADP(+) as acceptor(e.g., aldehyde dehydrogenase; Brouns et al., 2006). Enzymes belongingto E.C. 1.2.3 include oxidoreductases acting on the aldehyde or oxogroup of donors with oxygen as acceptor (e.g., aldehyde oxidase; Yamadaet al., 2015). Enzymes belonging to E.C. 1.2.5 include oxidoreductasesacting on the aldehyde or oxo group of donors with a quinone or similarcompound as acceptor (e.g., a dehydrogenase; Klein et al., 1994; Zhanget al., 2003).

In some implementations, the polypeptide that catalyzes reaction (b) maycomprise or consist of a lactaldehyde dehydrogenase (E.C. 1.2.1.22). Insome implementations, the lactaldehyde dehydrogenase may be encoded bythe gene aldA.

In some implementations, the expression of the polynucleotide encodingthe polypeptide that catalyzes reaction (b) may be placed under thecontrol of a constitutively active promoter.

In some implementations, expression of the polypeptide catalyzingreaction (c) may be deliberately controlled or attenuated to maintainthe intracellular levels of glycolaldehyde to sub-toxic levels (e.g.,via the use of a weak promoter, an inducible promoter, and/or low-copynumber plasmid).

(c) Conversion of Glycolate to Glyoxylate

In some implementations, the microorganisms described herein maycomprise a polynucleotide encoding a polypeptide catalyzing reaction(c), i.e., the conversion of glycolate to glyoxylate, wherein thepolypeptide comprises an enzyme of class E.C. 1.1.3.15, or a functionalvariant or fragment thereof.

Enzymes of class E.C. 1.1.3.15 include oxidoreductases acting on theCH—OH group of donors with oxygen as acceptor. In some implementations,the polypeptide that catalyzes reaction (c) may comprise or consist ofglycolate oxidase.

As indicated in FIG. 1 , in some implementations, the oxygen-dependentenzymatic conversion of glycolate to glyoxylate acts as anoxygen-dependent metabolic valve that enables the microorganism to (1)utilize glyoxylate for cell growth (biomass accumulation) at higheroxygen concentrations in a growth phase, and (2) allow glyoxylate toaccumulate under lower oxygen concentrations in a production phase.

In some implementations, the expression of the polynucleotide encodingthe polypeptide that catalyzes reaction (c) may be placed under thecontrol of a constitutively active promoter, an oxygen-sensitivepromoter, a pH-sensitive promoter, or an inducible promoter. In someimplementations, it may be advantageous to employ a strong and/orconstitutively active promoter in order to maintain intracellular levelsof glycolaldehyde to sub-toxic levels.

(d) Export of Glycolate Extracellularly

In some implementations, the microorganisms described herein maycomprise a polynucleotide that encodes a polypeptide that catalyzes orfacilitates reaction (d) the export of intracellular glycolate to theextracellular environment (e.g., into the fermentation broth). Forexample, some microorganisms may comprise a native glycolate exporter,and some microorganisms can be genetically modified to express anexogenous and/or heterologous glycolate exporter.

In some implementations, the polypeptide that catalyzes or facilitatesreaction (d) may be annotated as a glycolate permease such as glcA fromEscherichia coli, a formate permease such as focA from Escherichia coli,a lactate/glycolate symporter such as lldP from Escherichia coli orglycolate/glycerate transporter such as PLGG1 from Arabidopsis thalianaor a functional fragment or variant thereof.

In some implementations, it may be advantageous to express apolynucleotide encoding a fusion protein comprising enzymes thatcatalyze two or more reactions described herein (e.g., reactions (a) and(b), or (b) and (c)). Such fusion proteins are within the scope of thepresent description.

In some implementations, for example where glycolate is not the finalproduct, it may be advantageous for the microorganism to convertglycolate to a downstream product of interest such as polyglycolic acid,ethanolamine, or glycine. In such implementations, the microorganism maybe further genetically engineered to comprise one or polynucleotidesencoding one or more enzymes for converting glycolate to polyglycolicacid, ethanolamine, and/or glycine. The microorganism may then also befurther genetically engineered to express an exporter of polyglycolicacid, ethanolamine, or glycine that exports intracellular polyglycolicacid, ethanolamine, or glycine to the extracellular environment.

Various aspects, implementations, embodiments, features, examples, andexperiments regarding the present technology will be described infurther detail below.

Work, Findings & Experimentation

A detailed description of work, finding and experimentation that hasbeen done in the context of the present technology and innovations, isprovided below.

Brief Summary of Work and Findings

A considerable challenge in the development of bioprocesses forproducing chemicals and fuels has been the high costs of feedstockrelative to oil prices that make these processes uncompetitive withtheir conventional petrochemical counterparts. Hence, in the absence ofhigh oil prices for a foreseeable future, which was the main driver forwhite biotechnology, there has been a shift in the industry to insteadproduce higher value compounds such as fragrances for cosmetics. Yetstill, there is a need to address climate change and developbiotechnological approaches for producing large market, lower valuedchemicals and fuels. In this work, ethylene glycol, a novel feedstockthat has shown promise to address this challenge, was studied. An E.coli was engineered to consume ethylene glycol and as a case study, forchemical production, glycolate production was examined. At the testedconditions, one positive example fermentation performance led to theproduction of 10.4 g/L of glycolate after 112 hours of production time.The results clearly suggest that oxygen concentration is an importantfactor in assimilation of MEG as a substrate. It was also found that theuptake rates for ethylene glycol are sufficient to satisfy commercialbenchmarks for productivity and yield. Finally, the use of metabolicmodelling shed light on the intracellular distribution through thecentral metabolism implicating flux to 2-phosphoglycerate as the primaryroute for MEG assimilation. Overall, the work described herein suggeststhat ethylene glycol is a useful platform for commercial synthesis offuels and chemicals that may achieve economic parity with petrochemicalfeedstocks while sequestering carbon dioxide.

Introduction and Comments on Work

Biotechnological approaches to addressing climate change and the need tosequester carbon dioxide have focused on the development of microbialstrains engineered to produce chemicals and fuels derived from renewablesources of sugar. Despite the considerable success at engineering thesestrains, the lack of many successes at the commercial scale belies theimmense challenge in the financial viability of these technologies inthe face of low oil prices and expensive feedstock costs. In response,non-sugar feedstocks have been put forward as alternatives to competeefficiently with glucose based bioprocesses. For example, methane andsyngas fermentations are currently under intense study and are also thefocus of commercial development¹⁻³. Formate is another chemical that hasbeen suggested as a replacement for glucose since it can be producedfrom carbon dioxide and because of its inherent compatibility withbiological processes^(4,5). However, its utility as feedstock forbiological processes suffers from a number of drawbacks. The mostevident drawback is the absence of pathways for its assimilation in themetabolism of traditional workhorse organisms such as yeast or E. coli.The oxidized nature of the substrate also results in carbon loss toenable synthesis of NAD(P)H co-factors that support product and ATPformation, and the requirement for high transport rates into the cell toachieve productivities similar to glucose or xylose fermentation. Hence,while appealing, the technical challenges are numerous.

Nonetheless, this appeal arises from the fact that formic acid can begenerated electrochemically from CO₂. A one electron pair reduction ofone mole of CO₂ produces one mole of formic acid. However by tailoringthe catalyst and the reduction potential, multi-electron reduction canbe achieved and it is possible to produce a variety of different reducedcarbon species⁶. Biological processes have been used to produce many ofthese same chemicals that are typically produced by the petrochemicalindustry including 1-propanol, acetate, ethylene, etc⁷⁻⁹. The work,here, uses the observation that like formate, these other chemicals thatcan also derived by the electrochemical reduction of CO₂ are feasiblegrowth substrates for biological processes and this should merit theirconsideration as alternative feedstocks for bioprocesses.

In evaluating these substrates as potential replacements for glucose, itis important to recognize that many cannot be naturally catabolized bytraditional industrial workhorses. Hence, similar to formate, themetabolic engineering of substrate utilization pathways is preferred fordesired production performance. Additionally, many of the potentialreplacements are toxic and not compatible with bioprocesses. Others,while technically feasible as inputs to biological processes, sufferfrom poor faradic efficiency or poor selectivity in electrochemicalreactors¹⁰⁻¹³. Hence, after screening from a list of products that canbe generated electrochemically, it becomes apparent that only a few canbe realized as practical substitutes for glucose⁶. Finally, beyondtoxicity and efficiency which can be evaluated in a relativelystraightforward manner, evaluating the feasibility of a new substratefor bio-based chemical production can be obfuscated by how itsutilization is linked to the highly interconnected metabolic network.Indeed, refactoring large metabolic pathways into heterologous hosts hasproven challenging in the past¹⁴. One method that may help to explainwhy a new substrate performs poorly examines the metabolic pathway thatsupports a substrate for chemical production in relation to the cell'sentire metabolism.

In an earlier study²² this relationship was characterized by calculatingthe interactions between two competing objectives of cellular systems,growth and chemical production. The theory laid out how the underlyingnetwork structure gives way to growth independent chemical production.That relationship was captured by a mathematical framework usingelementary flux modes to measure the interconnectedness of the cellsystem and the desired objectives. Hence, a metric was defined tomeasure the orthogonality of the chemical production pathways withrespect to the biomass production.

It was found that the organization of ideal metabolic structuresdesigned to minimize cell-wide interactions had a characteristicbranched topology. This type of orthogonal structure could be exploitedfor two stage fermentation. Furthermore, an important finding from thatstudy was glucose, while a common substrate for industrial fermentation,is not ideally suited for chemical production objectives due to thesignificant overlap between the pathways for biomass synthesis andchemical production. Instead, substrate selection should be based on thechemical targeted for production. Among the various substrates andproducts, it was identified that ethylene glycol was a highly promisingsubstrate for orthogonal production of a variety of chemicals because itminimized the interactions between biomass and chemical producingpathways.

Therefore, among the variety of different chemicals that can be producedelectrochemically, ethylene glycol is a promising, unconventionalfeedstock. It is produced today primarily by the petrochemical industryfrom ethylene. However, a process for making ethylene glycol from CO₂has shown early promise, and is currently the focus of industrial scaleup. In this regard, its utilization as a feedstock for biologicalprocesses is important because it can serve as a replacement for glucosein the modern bioprocess.

In the present work, an E. coli was engineered and characterized as abiocatalyst capable of consuming ethylene glycol as a carbon source, andits application as a novel substrate for industrial bioprocesses wasexplored. This platform for growth and chemical production was thenapplied to a case study for glycolic acid production. This case studyattempts to validate an orthogonal approach for chemical production,relating the network topology and two-stage fermentation. Conventionalapproaches to glycolic acid in E. coli have instead focused on usingglucose as the substrate, and implementing genetic strategies thatcouple production to growth. Several studies have been published thathave examined glycolic acid production from glucose^(15,16) andxylose¹⁷⁻¹⁹. The highest of these reports achieves titers of 56.44 g/Land a yield of 0.52 g/g²⁰. To our knowledge, only three studies haveexamined ethylene glycol conversion to glycolic acid as abiotransformation²¹⁻²³. However, in this work the metabolism and growthphysiology of E. coli growing on ethylene glycol were thoroughlycharacterized. It was found that while growth rate is markedly slowrelative to growth on glucose with a doubling time of 3.85 hours onethylene glycol, that the substrate uptake rate is sufficiently high atup to 5 mmol/gDW-h to be relevant for industrial production. Glycolate,which required micro-aerobic conditions, reached titres of 10.4 g/L at amaximum theoretical yield of 66%.

Overall, it was found that understanding the growth characteristics ofthe cell and a model on glycolate production shows that using ethyleneglycol has potential for replacing glucose in industrial bioprocesses inapplications where CO₂ streams and renewable electricity are available.

Materials and Methods

Media and Cultivation Conditions

Cells were grown using lysogeny broth (LB) as per manufacturer'sinstructions (Bioshop, Burlington, ON) for all strain construction andfermentation pre-cultures. When characterizing strains, cells were grownunder M9 minimal media with the following compositions: 1.0 g/L NH4Cl,3.0 g/L KH₂PO₄, 6.8 g/L Na2HPO4, 0.50 g/L NaCl. Supplements of yeastextract at 2 g/L were added to minimal media. Ethylene glycol was usedas the carbon source as concentrations described in the text. IPTG wasused at a concentration of 1 mM when necessary. A trace metal solutionwas prepared according to the following composition prepared in 0.1 MHCl per litre and added at a concentration of 1/1000: 1.6 g FeCl₃, 0.2 gCoCl₂·6 H₂O, 0.1 g CuCl₂, 0.2 g ZnCl₂·4H2O, 0.2 g NaMoO₄, 0.05 g H₃BO₃.1 M MgSO4 and 1 M CaCl₂ was also added to the media at a concentrationof 1/500 and 1/10,000, respectively. For all cultures, carbenicillin wasadded as appropriate at 100 μg/mL. Cells were grown in 250 mLshake-flasks for all characterization experiments and in bioreactors asdescribed.

Culturing Techniques in Reactors

Pre-cultures were grown in LB rich media in 10 mL test tube culturesovernight and transferred fresh shake-flaks containing LB, 1 mM IPTG and10 g/L ethylene glycol. After 24 hours, these cells were harvested bycentrifugation, re-suspended in 2 mL of residual supernatant and used asinoculum for bioreactor or minimal media shake-flasks forcharacterization at 37° C.

Applikon MiniBio500 fermentation vessels were used for cultivatingstrains in bioreactors. Dissolved oxygen and pH probes were used inaccordance with the manufacturers operating guidelines. M9 minimal mediawas used for cultivation in the bioreactor. pH was maintained at 7 withthe addition of 3N KOH. Growth conditions were maintained at 37° C.Dissolved oxygen was maintained as described in the text. Flowrate wascontrolled as described using a Books Instruments mass flow controllers(GF Series) and gas was analyzed using Thermo Scientific™ Sentinel dBmass spectrometer for online gas measurement.

Analytical Methods

Analysis of fermentation production was measured via high performanceliquid chromatography (HPLC). A Bio-rad HPX-87H organic acids columnwith 5 mM H₂SO₄ as the eluent and a flowrate of 0.4 mL/min at 50° C. wasused. Organic acids were detected at 210 nm. Cell densities of thecultures were determined by measuring optical density at 600 nm (GENESYS20 Visible Spectrophotometer). Cell density samples were diluted asnecessary so as to fall within the linear range. A differentialrefractive index detector (Agilent, Santa Clara, Calif.) was used foranalyte detection and quantification. Yields were calculated between twotime points, whereas the cumulative yield was calculated between theinitial and final measurements.

Plasmids and Strains

fucO and aldA were cloned from E. coli MG1655 genomic DNA and assembledusing Gibson Assembly²⁴ into a pTrc99a vector. Ribosome-binding site(RBS) sequences were placed onto the overhang of the forward primer.AACAAAATGAGGAGGTACTGAG was the RBS sequence used in front of aldA.AAGTTAAGAGGCAAGA was the RBS sequence used in front of fucO. The Trcpromoter was used to drive expression. Wild-type strains of E. coliMG1655 were obtained from the Coli Genetic Stock Centre (Yale).

Flux Balance Analysis

Flux balance analysis (FBA) was performed using MATLAB R2015a installedwith COBRA 2.0 toolbox and using the GLPK linear solver (GNU Project).The genome scale model iAF1260 was used to perform all modelling. TheATP maintenance reaction was left unchanged at a value of 8.9mmol/gDW-h. The model was modified by adding a reaction for convertingethylene glycol to glycolaldehyde using NAD cofactors. Transport ofethylene glycol was modelled as free diffusion and no protontranslocation was included as part of its exchange reaction. Initialcharacterization ofthe cell to model the respiratory quotient was onlyconstrained by its substrate uptake rate which was measured at 5mmol/gDW-h. More detailed intracellular flux data was extrapolated byconstraining substrate uptake rate as well as glycolate production ratesand oxygen uptake rates as determined by analysis of the off-gas fromthe process mass-spec during bioreactor cultivation.

Results

Ethylene Glycol is a Preferred Substrate Over Formate

In an earlier study, orthogonality was identified as a metric to assessand design efficient metabolic networks for the production of chemicals.That study defined orthogonality as a quantitative measure of theinterconnectedness between pathways that produce a target chemical andbiomass. The principal focus of that work was to examine how metabolicpathway organization influences chemical production. In this firstsection, that methodology was applied to compare formate and ethyleneglycol utilization, both of which can be synthesized electrochemically.Also assessed was the specific role that substrate selection has on fivedifferent chemicals that are important to industry found in Table 1.This analysis allows us to implicitly account for metabolic constraintssuch as redox and ATP. Glycolic acid showed the highest orthogonalityscore between all the substrate product pairs, and hence was selected asthe demonstration product for production from ethylene glycol.

TABLE 1 Succinate Ethanol Glycolate 2,3-Butanediol Score Yield ScoreYield Score Yield Score Yield Formate 0.47 0.29 0.50 0.14 0.48 0.33 0.490.18 Ethylene 0.54 0.95 0.61 0.62 0.67 1.22 0.66 0.66 glycol Glucose0.41 1.12 0.44 0.51 0.41 0.85 0.47 0.50 Xylose 0.36 1.11 0.36 0.65 0.340.65 0.40 0.64

Table 1 shows the orthogonality score for these chemicals using ethyleneglycol and formate as carbon sources. Glucose and xylose are alsoincluded in the calculations as they provide a reference against theconventional bio-process. For all chemicals, the orthogonality score islarger for ethylene glycol than formate and less substrate is requiredto produce the same quantity of product as well. Table 1 thus relates toyield and orthogonality metrics for chemical production from differentsubstrates. The orthogonality scores for various products are showncomparing two substrates that can be generated electrochemically againstconventionally used substrates by their natural pathways. Formate hasorthogonality scores similar to many sugar-consuming pathways,indicating a relatively complex and inter-connectedness for itsutilization. The highest scores are those for ethylene glycol withyields that are better than the sugars glucose and xylose. Yield isgiven as g of product per g of substrate.

The orthogonality metric is a mathematical measure of the set ofinteractions that each substrate assimilation pathway has to the cellcomponents outside their pathways. Hence, it implicitly measures thebiological complexity one might expect to ensure that the biomolecularmachinery of that pathway can concurrently function within the cell'snatural metabolism to support biological and chemical productionobjectives.

Analysis of the metabolism of formate shows its low score arises fromits low degree of reduction which requires flux through the TCA cycle togenerate the necessary reducing equivalents for growth and energy,irrespective of what chemical is produced. The low degree of reductionis also the reason for low product yields. Hence, this line of networkanalysis suggests ethylene glycol is a superior substrate to formate inE. coli. Given higher scores for ethylene glycol utilization, it wasresolved that ethylene glycol utilization was promising and theseresults were compared to sugar metabolism in E. coli for glycolic acidproduction.

Glycolate is an alpha-hydroxyacid used in the synthesis of a variety ofdifferent plastics and polymers, cosmetics and industrial detergents.Currently, metabolic engineering has established routes to glycolic acidfrom glucose and from xylose. Theoretical yields have been dependent onboth the substrate selected as well as the biosynthetic pathway used forproduction. Examples of glycolate production from glucose in literaturehas primarily been demonstrated by the activation of the glyoxylateshunt.

Regarding FIG. 1 , it is noted that glycolate can be produced by avariety of different substrates. Ethylene glycol conversion to glycolateis shown in (A). The two most commonly studied substrates for productionare xylose (B) and glucose (C). To efficiently produce glycolate fromglucose or xylose, genetic interventions are required to the centralmetabolism to couple growth and glycolate synthesis. The focus of thisstudy examines ethylene glycol consumption. Limiting oxygen provides amechanism to permit glycolate accumulation. Under fully aerobicconditions, glycolate is converted to glyoxylate and channeled to thecentral metabolism for growth via the glycerate metabolism. Under oxygenlimiting conditions, glycolate accumulates.

FIG. 1 shows glycolate production from three different pathways.Production from glucose is highly coupled to biomass synthesis, andexhibits the lowest orthogonality score, 0.41. Glycolate production fromxylose has also been demonstrated by the use of a synthetic pathway forxylose assimilation in E. coli. While this pathway fits partly into anorthogonal criterion for glycolate production, the concomitantproduction of pyruvate for every mole of glycolate requires the use ofthe cells highly interconnected glyoxylate cycle to reach theoreticalyields. The orthogonality score, for this reason, is comparativelysmaller. The largest orthogonality score of 0.67 was found for the casewhere ethylene glycol served as a substrate. Bioconversion of ethyleneglycol to glycolate fits into the ideal network architecture thatfollows a branched pathway. Under oxygen limiting conditions, thereaction that consumes glycolate, catalyzed by glycolate oxidase, can belimited, and the cell can accumulate glycolate. These results show thatethylene glycol as a substrate is more orthogonal than traditionalsubstrates and hence suitable for validating as a concept of orthogonalpathways based design.

Ethylene Glycol Utilization by E. coli

There exist pathways in nature that allow microorganisms to consumeethylene glycol as a carbon source²⁵⁻²⁸. While not commonly reported inmetabolic engineering applications, these organisms use one of two typesof metabolic pathways. The first pathway utilizes a diol-dehydrataseresulting in the dehydration of ethylene glycol to acetaldehyde.Acetaldehyde is then activated to acetyl-Coa by an acetaldehydedehydrogenase enzyme which provides the cell with the key precursormetabolite to support growth via the TCA cycle and gluconeogenicpathways. The production of one mole of acetyl-Coa from one mole ofethylene glycol concomitantly produces one NADH. This pathway is mostcommonly found in some Clostridium species and a few other anaerobicorganisms owing to the oxygen sensitivity of thediol-dehydratase^(25,27). The second mode of ethylene glycol degradationutilizes a pathway wherein ethylene glycol is successively oxidizedusing nicotinamide cofactors and oxygen to produce glyoxylate.Glyoxylate which is a gluconeogenic carbon substrate, can then be usedas the growth metabolite as it enters lower glycolysis at the2-phosphoglycerate node as well as the TCA cycle via the glyoxylateshunt.

Wildtype E. coli MG1655 cannot naturally grow on or degrade ethyleneglycol. However, it is possible to select for this strain, and to ourknowledge, only one study has ever reported ethylene glycol utilizationby E. coli. ²⁹ That strain was selected from derivatives of propyleneglycol utilizing mutants. Researchers identified increased activities ofglycolate oxidase, glycolaldehyde dehydrogenase and propanedioloxidoreductase as the necessary components required for itsassimilation. More generally, a survey of the literature shows thatenzyme promiscuity is a relevant element of the utilization ofalcohols^(22,23). In this specific case, enzymes regarded as beingimportant for propanediol or even glycerol utilization across manyorganisms have shown activity on ethylene glycol and are regarded as thekey methods for degradation, irrespective of the dehydratase route orthe oxidative route via glyoxylate²⁶⁻²⁸. Hence, in this study, toengineer E. coli the native gene fucO and aldA that have beenestablished as key enzymes supporting propanediol utilization in E.coli, were overexpressed. Since FucO has previously been shown to besensitive to oxygen via metal catalyzed oxidation that results in theinactivation of Fe²⁺ dependent propanediol oxidoreductases, two variantsof the pathway to consume ethylene glycol were designed. In variant 1(strain LMSE11), the mutated version of fucO was used wherein I7L andL8V based on earlier mutagenesis studies'. In the second variant (strainLMSE12), L8M was used because it was also suggested to play a role inalleviating metal catalyzed oxidation (MCO) toxicity in propanediolassimilation by E. coli. Both variants had the same ribosome bindingsite and trc promoter upstream of the start codon.

Cells were grown aerobically in M9 minimal media with ˜10 g/L ethyleneglycol, supplemented with 0.2% yeast extract in 250 mL shake flasks.Fermentation profiles between the two strains constructed were markedlydifferent. LMSE11 completely consumed ethylene glycol in 47 hours whileLMSE12 had consumed only ˜10% ofthe initial substrate in same timeperiod with 10 g/L as residual MEG. These results are shown in FIG. 2 .

FIG. 2 illustrates cell growth curves and their substrate consumptionprofiles for the strains constructed in this study. The oxygen variantsof fucO showed a marked difference in growth rate and substrateutilization in shake-flask experiments. Ethylene glycol consumption isshown by the dashed lines and OD₆₀₀ is depicted by the solid lines.Yellow (light) shows strain LMSE11 while green shows LMSE12. Error barsindicate standard deviation of triplicate experiments.

Growth yield for LMSE11 was calculated to be 0.28 gDW/g MEG. Fluxbalance analysis via in silico simulations of the core model of E. colirevealed the theoretical yield to be 0.35 gDW/g MEG. These resultsseemed to be in reasonable agreement with theoretical yields for biomasssynthesis, suggesting that two genes are sufficient to efficientlyconvert ethylene glycol to biomass using E. coli's natural biosyntheticpathways. The substrate uptake rate in shake-flasks was determined to be5 mmol/gDW-h. The experimental growth rate was calculated to be 0.18 h⁻¹corresponding to a 3.85 hour doubling time. FIG. 2 shows the growthcurve and substrate utilization of for both variants. LMSE12 consumedsubstantially less ethylene glycol and had residual ethylene glycolconcentrations just under 10 g/L in the same time period.

Analysis of the fermentation media by HPLC showed the absence offermentation products like acetate or lactate, and the intermediatemetabolites glycolaldehyde and glycolate. However, since LMSE11 showedhigher utilization rates, it was decided to pursue that variant further.

Orthogonal Production of Glycolate by E. coli

Having established ethylene glycol consumption by an engineered strainof E. coli, the use of ethylene glycol as an orthogonal substrate forthe production of glycolic acid was explored. E. coli strain LMSE11 wasgrown in bioreactors with minimal media, supplemented with yeast extractat 2 g/L and sparged with air to maintain oxygen at 1 v/vm (300 mL/min).These conditions ensured that oxygen saturation above 50%. Cells wereinitially grown overnight for 18 hours for growth in LB rich mediasupplemented with ethylene glycol and induced with IPTG. After overnightgrowth, they were centrifuged, washed and suspended in minimal media andinoculated to bioreactors at an OD˜0.4 (approx. 0.23 gDW/L). Thebioreactors contained 1 mM IPTG to maintain induced expression of MEGutilization genes to support biomass.

At 20 hours, the aeration was reduced to 150 mL/min (0.5 v/vm) and 50mL/min (0.16 v/vm) to simulate high and low aeration rates, and theimpeller agitation was dropped to 500 rpm. It was observed that cellgrowth continued until approximately 40 hours reaching approximately 5gDW/L at which point cells in both reactors appeared to reach astationary phase. Production of glycolate, however, was continued for 30hours more after the beginning of stationary phase at which point thefermentation was stopped. Cells grown at a higher rate of aerationaccumulated more glycolate by the end of the batch. The final glycolatetitres for the two treatments were 2.5 g/L and 4.1 g/L. Using fluxbalance analysis to approximate carbon loss from respiration andaccounting for cell growth and other products, it was possible to closethe carbon balance at 83% and 88%, respectively. Average mass yield forglycolate on MEG measured during the production phase was 0.18 g/g and0.32 g/g. FIG. 3 illustrates results of these experiments.

Referring to FIG. 3 , influence of aeration on glycolate production isillustrated. To assess the impact of oxygen transfer in bioreactors,cells were grown under two aeration rates during the micro-aerobic phaseof the fermentation. (Top) High aeration had a flow rate of 150 mL/min.(Bottom) Low aeration was characterized by flow at 50 mL/min.Experiments were conducted in duplicate. Error bars indicate range ofthe measured values.

Counter-intuitively, the lower aeration led to lower glycolate titerseven though FucO in the MEG utilization pathway was expected to besensitive to higher oxygen levels. However, this result can be explainedby the fact that oxygen is required for the regeneration of NAD which isa substrate for the MEG utilization pathway. Hence, lower oxygenconcentrations could lead to lowered flux through this pathway resultingin lower titers. These results suggest a trade-off between the oxygensensitivity on the one hand and the requirement for oxygen as asubstrate in the pathway. Next, it was desired to analyze the role ofoxygen further using metabolic modeling and by increasing the aerationrate even further to see if glycolate production could be enhanced.

Dissolved Oxygen and Control Over Metabolism

To gain further insight into control of the cell's metabolism usingoxygen and refine our approach to glycolate production, flux balanceanalysis (FBA) was used to simulate the intracellular flux through thecentral metabolism at 5 mmol/gDW-h which was determined with theshake-flask experiments. The simulations were constrained using thesubstrate uptake rate to approximate E. coli growth during the earlyexponential growth phase measured in shake flasks. The ATP maintenanceflux was approximated at 8.9 mmol/gDW-h, a value experimentally used forglucose metabolism. The simulated flux distributions revealed a highlyreorganized central metabolism of E. coli using gluconeognic pathways.

Under oxygen limiting conditions FBA predicts the observed fermentativecell behavior and glycolate accumulation. Then, this observation wasexplored further by modelling the production of the glycolate (as yield)by the cell and its respiratory quotient as a function of the oxygenuptake rate. This analysis allowed us to implicitly correlate theflowrate ofn air into the reactor to the metabolite production yieldssince the specific oxygen uptake is a function of air intake. FIG. 4shows that the increase in glycolate yield and the onset of fermentationas oxygen uptake rate is reduced. These yields correlate with therespiratory quotient (RQ) that also decreases at a lower oxygen flux andincreases with increasing oxygen flux before it levels off at saturatingconditions. These results suggest that RQ is an important variable thatcan be monitored and controlled to optimize for glycolate production inreal-time. Hence, this approach was used to control glycolate productionin subsequent runs by ensuring that there was sufficient aeration.

Regarding FIG. 4 , a graph related to metabolic modelling of glycolateproduction is shown, where glycolate yield (glycolate, blue), therespiratory quotient (RQ, green) and the substrate specific productivity(SSP, red) were modelled using FBA. Glycolate production begins at theonset of oxygen limitation which occurs at approximately 8 mmol/gDW-h ofoxygen. At greater values, the RQ plateaus as sufficient oxygen asavailable for complete respiration and FBA predicts no glycolateaccumulation. The grey bar indicates the values at which RQ wascontrolled experimentally during the production phase in later batches.

Glycolate Production and Fed Batch Strategy

Finally, given that it was possible to produce glycolate, furtherexperiments were performed to attempt to improve glycolate productionyield and increase titres. Based on what was learned from the initialfermentations, it was sought to increase the glycolate production phaseand reduce the biomass production phase. This was achieved by increasingthe aeration rate to 2 v/vm (600 mL/min) during the growth phase of thebatch to prevent glycolate accumulation and divert as much flux towardsbiomass. In the second phase, the aeration rate was dropped to 100mL/min. Results of this strategy are shown in the FIG. 5A. Finalglycolate titres reached 6.8 g/L after approximately 70 hours productiontime with an initial production phase biomass concentration ofapproximately 4 gDW/L, corresponding to an average productivity 0.1g/L-h or approximately 0.32 mmol/gDW-h. The initial yield of glycolatewas 0.92 g/g after the first sample was taken, however, the cumulativeyield decreased during the production course of the batch with the finaloverall production yield of 0.75 g/g or 61% of theoretical.

It was observed from these conditions that while significantly moreproduct was produced at a higher yield, the cells took much longer toreach a concentration appropriate for a production phase. Whereas whenthe aeration rate was 1 v/vm in earlier batch, the cells reached aconcentration of 4 gDW/L within 30 hours. However, at 2 v/vm it tookalmost 70 hours to reach the same concentration. It was hypothesized thelonger time to reach a higher OD was likely due to increased dissolvedoxygen levels and faster oxygen mass transfer rates to the cells duringearly exponential phase. Given the sensitivity of FucO to oxygen, ineven the mutant variant, these two factors likely created an oxygentoxicity on the cells resulting from the inactivation of these proteinsby metal-catalyzed oxidation and placing a high metabolic burden on thecell in regards to high protein demand without a sufficient means toutilize ethylene glycol as a carbon source.

Oxygen requirements is also one of the factors that affects theindustrial production of biochemicals since it is a key component ofoperating costs which are determined by the energy inputs. One of thesignificant energy inputs for a process is the energy needed to aerate abioreactor. In an earlier experiment, it was found thatcounter-intuitively, a higher aeration resulted in higher glycolatetitres at a higher yield but that high aeration also retards cellgrowth. From a process perspective, it is desirable to operate a reactorat a lower flow rate. Building on all these earlier studies and thevarious competing objectives it was attempted produce glycolate at ahigh titre but at a lower aeration rate. Hence, cells were grown under aconstant aeration 0.16 v/vm (50 mL/min), but during the productionphase, the impeller speed in the reactor was dropped until the RQ, asmeasured by the online mass-spec read ˜0.4. The working hypothesis basedon FBA simulations was that this would achieve a yield greater than 0.4mol/mol and place the production phase in near its maximum substratespecific productivity. The shaded region in FIG. 4 shows the range ofthe RQ measured during the course of the production phase as determinedby three standard deviations from the average value. The average RQ wasmeasured to be 0.37. The results of this experiment are shown in FIG.5B. It was possible to reduce the biomass production phase to 26 hours,and produce 10.4 g/L of glycolate over a 112 hours production phase. Theoverall yield was determined to be 0.8 g/g from ethylene glycolcorresponding to a molar yield of 0.66 mol/mol. The productivity wascomparable to earlier experiment at 0.1 g/L-h. These experimentalresults were in line with and correlated well with FBA predictions forusing RQ as a control variable. As the batch entered the glycolateproduction phase, it was observed a drop in the RQ. However, themeasured RQ value of 0.37 corresponded to a production yield of 0.66mol/mol—higher than the expected yield of 0.40 mol/mol. The resultsimply that while the general agreement between experimental data and FBAsimulations are useful in establishing a control mechanism forfermentation on ethylene glycol, further optimization of modelparameters is required to accurately predict physiological response tothe environmental conditions. In particular, it was found that substrateuptake rate was reduced substantially in vivo however (approximately 0.7mmol/gDW-h), which was not accurately captured by the FBA models (at 3.5mmol/gDW-h).

Regarding FIGS. 5A and 5B, which relate to fermentation profiles for fedbatch strategies, fed batch studies were conducted to assess the longterm stability of the production phase. The production phase isseparated from the growth phase by grey shading (A) Shows bioreactorconditions at 2 v/vm during the growth phase and 0.33 v/vm during theproduction phase at a cell density corresponding to 4 gDW/L. (B) Cellswere grown at 0.167 v/vm air flow rate into the bioreactor with anaverage stationary phase cell density at 2.5 gDW/L. Cells were capableof robust glycolate production for well over 100 hours in the productionphase.

Metabolic Flux Analysis Using E. coli Model

To gain insight into the intracellular fluxes of the cell, mass spec andHPLC data were used to constrain a genome scale model of E. coli andperform flux balance analysis. The model was then used to estimate theintracellular fluxes under ethylene glycol growth conditions to gaininsight into the cellular metabolism. It was determined that ethyleneglycol enters the metabolism at the glyoxylate node (FIG. 6A). 70% ofthe glyoxylate production flux is channeled towards 2-phosphoglycerate(2PG) under aerobic conditions which enters lower glycolysis. Theremaining glyoxylate is used to generate malate via malate synthase. Itappears from the simulations that majority of the malate and 2PGgenerated by these pathways ends up in the TCA cycle. As a percentage,65% of the total carbon entering the cell as ethylene glycol getschanneled into acetyl-coa. Conversely, about a fifth of the total carbonget channeled by gluconeogenic pathways towards upper glycolysis and thepentose phosphate pathways.

During the growth phase it was also observed small amounts of glycolate.The accumulation of glycolate suggested insufficient oxygen and thus thepossibility that anaerobic pathways in the cell may be induced.

Indeed trace amounts of formate were detected as peaks in the HPLCchromatogram.

Given that the 2PG pathway that assimilates ethylene glycol results incarbon loss via the tartronate semi-aldehyde carboligase step,simulations were performed to determine whether the glyoxylate cycle wassufficient for supporting cell growth by removing the reaction glyck2(glycerate kinase) from the model. Removal of glyoxylate carboligasefrom the genome scale model showed a 50% decrease in the in silicogrowth rate. In contrast, experimental work on gene deletions in thesame pathway show that it abolishes growth on glycolate. To reconcilethese differences, the genome scale model was analyzed to determine thespecific reactions that support cell growth. It was found that withoutglyoxylate carboligase, cell growth could theoretically be supported bythe threonine pathway where oxaloacetate is converted to serine,homoserine and threonine. Threonine aldolase is capable of cleaving theamino acid to glycine for growth, and acetaldehyde for providing theacetyl-Coa necessary to replenish the acetyl-Coa that is consumed bymalate synthase. Hence, it is the threonine metabolism generated fromoxaloacetate that provides the route to support biomass in silico. Thispathway converts acetyl-Coa to glycine. However, it is unlikely thatthese enzymes are expressed in sufficient quantities to carry enoughflux to support growth. Hence, the primary role of the secondary malatesynthase pathway and flux split in glyoxylate metabolism between theglyck2 and mals (malate synthase) reactions seems to be to replenish theTCA cycle intermediates as opposed to assimilating ethylene glycol.

A similar methodology was applied to determine the intracellular fluxdistribution under the micro-aerobic conditions. During the glycolateproduction phase (FIG. 4-5B), oxygen flowrate into the bioreactors waslimited to create a micro-aerobic environment. The resulting drop inoxygen concentration affected the metabolic flux distribution. The mostnotable change was a reduction in the substrate uptake rate of ethyleneglycol to ˜0.7 mmol/gDW-hr, a quarter of what was observed duringaerobic growth. Secondly, in silico simulations predicted reducedglyoxylate utilization through malate synthase and instead majority ofthe flux was diverted towards the TCA cycle through 2PG. Whereas themolar ratio of flux through lower glycolysis versus malate synthase wasalmost 1:1 under aerobic conditions, it was estimated to be 30:1 undermicro-aerobic conditions. The decrease in the substrate uptake, it couldbe speculated, is likely caused by a lower oxidation rate of NADH byoxygen leading to an accumulation of reduced NAD co-factors and leavingfewer oxidized molecules available for ethylene glycol catabolism. Theproduction of acetate in the metabolism is a characteristic of over-flowmetabolism associated with fermentative metabolism. Trace amounts offormate, produced by pyruvate formate lyase which is transcriptionallycontrolled by oxygen is consistent with other studies showing activationof anaerobic pathways in the transition to a fermentative metabolism.

Regarding FIG. 6 , it shows flux distribution of the metabolism and keyenzymes in the pathway. Part (A) shows the estimated intracellular fluxdistribution under aerobic conditions. Part (B) shows under oxygenlimiting conditions, the metabolic model estimates ethylene glycol fluxethylene glycol is primarily converted to glycolate. Values in bracketsrepresent upper and lower values obtained from flux variabilityanalysis. The flux ranges provide an estimate of the error in thereaction fluxes based on the constraints imposed for the abovesimulation. In this case, the relatively narrow ranges on theestimations are useful to attribute a physiologically meaningfulinterpretation to the data.

Use of Alternative Ethylene Glycol Oxidizing Enzymes Enables EthyleneGlycol Consumption

Whereas it is shown herein that fucO and aldA can impart onto E. colithe ability for assimilating ethylene glycol as a carbon source, it isdesired in some instances that an alternative to fucO be used when cellsare grown under conditions of high aeration. This is because the FucOenzyme (and many other enzymes that use iron as a cofactor) contains aniron-sulphur cluster that is prone to inactivation in the presence ofoxygen. Hence, several candidate enzymes that use zinc as a cofactorwere tested and validated in vitro for their ability convert ethyleneglycol to glycolaldehyde. One of the enzymes that was validated in vitro(the Gox0313 gene from Gluconobacter oxydans) was expressed inEscherichia coli MG1655, along with aldA on a low copy plasmid (p15Aorigin of replication). Cells were grown in M9 minimal mediasupplemented with 20 g/L ethylene glycol and 0.1% yeast extract in 250mL shake flasks. Fermentation profiles of the strains confirm theability to use Gox0313 for assimilating ethylene glycol. In comparison,the wild-type strain showed little growth resulting from the yeastextract present in the growth medium (see FIG. 9 ). In analyzing theresults of this experiment, we observe it took 41 hours for cells to gofrom an OD600 of ˜0.4 to ˜9.9. By comparison, in an earlier experimentwhere a fucO variant with reduced sensitivity to oxygen was used, weobserved that it took approximately 45 hours to go from an OD600 of ˜0.4to ˜6.2. Thus, the Gox0313 containing variants reached a higher totalbiomass concentration over a shorter period of time relative to the fucOvariants. Hence, these results suggest that this Zn containing enzyme issuperior in its ability to support growth and ethylene glycolutilization over the fucO variants that contain an Fe co-factor.

Discussion

Conventional approaches to the bio-based production of chemicals haverelied on using glucose, and more recently xylose as feedstocks. Yetmicroorganisms tend to be very diverse in their ability to metabolizedifferent carbon sources. In this work, the use of ethylene glycol as asubstrate to replace glucose in bioprocesses for growth and chemicalproduction was proposed and examined. Counter to other studies, manypertaining to the synthesis of ethylene glycol from glucose, ourmotivation for studying ethylene glycol as a substrate stems from thefact that it can also be derived from CO₂ ^(6,31). Hence, itsconsideration as a feedstock that can potentially sequester carbon andlower greenhouse gas emissions is akin to studies examining syngasfermentation of formate utilization.

To assess ethylene glycol utilization in the context of biochemicalproduction, glycolic acid production was examined. Glycolic acid is analphahydroxy acid used in cosmetics and polymer applications. Theresults from our study allows us to conclude that ethylene glycol is asuitable platform for growth and highly efficient for producing glycolicacid. More generally it was found that with further metabolicengineering, ethylene glycol could be used to produce alcohols and otherorganic acids that are typically produced during fermentativemetabolism. This capability, it is believed, can have an impact inindustrial biotechnology. Elaboration on these findings is provided byexamining three specific areas.

Consideration of ethylene glycol as a substrate can be driven bychallenges related to the utilization of non-native substrates in E.coli. These interactions, which were described earlier as orthogonality,help to identify pathways with high and low degrees of interactions.Computationally, it was found that ethylene glycol exhibits a lowerlevel of interactions than many natural and some synthetic pathwayswhich can make it a more robust substrate than substrates such asformate or methanol. Hence, these interactions provided a rational basisfor selecting and engineering a novel substrate utilizing pathway intoE. coli. This work demonstrates the first de novo design of orthogonalpathway for metabolic engineering based on an orthogonality metric.

The results demonstrate the applicability of E. coli to use a new andnovel substrate that has never been considered as a potential feedstock.Initial characterization of the cell growth determined that thesubstrate uptake rate was approximately 5 mmol/gDW-h. At typical celldensities for industrial processes (10-100 g/L)²⁴, this corresponds tonet flux of 3-30 g/L-h, well above the required 3-4 g/L-h productivityfor growth independent production typically needed²⁵. Furthercharacterization of these strains led us to determine that there wassome oxygen sensitivity, especially during early exponential phase. Itis believed that these are likely caused by metal catalyzed oxidation ofFucO in the presence of excess aeration and could be addressed by usingO₂-tolerant Zn²⁺-dependent variants.

An important observation made during the course of these experiments wasa reduction in the substrate uptake rate during oxygen limitingconditions. It is believed that the oxygen limitation results inincreased

NADH pools leading to a decrease in the rates of reaction catalyzed byfucO and aldA. This change in the rates had a net effect of lowering theflux of ethylene glycol into the cell. This finding necessitates afurther study of cellular physiology under ethylene glycol utilizationso as to understand the trade-off in yield and productivity as afunction of the dissolved oxygen feeding in the bioreactor. For example,whereas increases were found in overall glycolate titres at 150 mL/minrelative to 50 mL/min further, on-line monitoring in the fed-batchstudies via maintaining a target respiratory quotient helped to increaseproduct yields and titres at 50 mL/min relative to the earlierexperimental conditions at 150 mL/min. Hence, optimization of aerationin the bioreactor would substantially improve economic performance, bothin terms of product formation but also in terms of the absolute cost ofaeration. For example, the operating conditions of the experiment inthis bioreactor, correspond to a k_(L)a of 120 h⁻¹. Typical jet loopbioreactors²⁶ are capable of delivering this design constraint at a masstransfer power of 3 kW/m³. Therefore, a typical reactor that is 350 m³would consume 1000 kW of power or 160,000 kWh over the course of atypical fermentation. This requirement corresponds to an energy cost (at$0.10/kWh) of over $15,000 which represents 20%, a substantial fraction,of the final cost of the product at 100 g/L at $2/kg in a typical350,000 L fermenter. Hence, the importance of optimizing processconditions through genetic engineering is important to its financialviability. Further work entailing a more detailed study of the oxygentransfer and glycolate titres is expected to more accurately determinethe optimum conditions.

Further computational modelling allowed us to infer ratios of key branchpoints within the metabolism and identified glyoxylate carboligase asthe central pathway for assimilating ethylene glycol, with malatesynthase playing a relatively small role in its assimilation. Results ofthis modeling also showed that the much of the NADPH redox requirementsfor cell growth were surprisingly obtained through the pentose phosphatepathway and relatively little from the anaplerotic NADP dependent malicenzyme, as might be initially expected. It was also observed smallamounts of acetate and trace amounts of ethanol in the fermentationmedia during microaerobic glycolate production phase. FBA modellingresults predicted ethanol production during microaerobic conditions, butfailed to predict acetate production, without the adequate constraints.The observation of acetate and ethanol in the fermentation medium,typical products of anaerobic growth suggest that microaerobicconditions may permit ethylene glycol as a suitable feedstock for theproduction of other anaerobic products despite its requirement foroxygen. Finally, by extending the observations from flux balanceanalysis, it was possible to use a process mass spec to measure inreal-time the respiratory quotient and by use of a simple model, andshow its applicability as parameter to control glycolic acid productionduring the course of the fermentation. This may open new opportunitiesfor producing a variety of products using ethylene glycol as a feedstockprovided the oxygen mass transfer rate can be efficiently controlled.

The results described herein establish a framework for future productionof chemicals in E. coli using ethylene glycol as a substrate. Describedherein, for the first time, is the successful production of glycolicacid from ethylene glycol using the substrate as a feedstock for growthand for production. A bioprocess based on ethylene glycol as a feedstockcan have important implications and applications in the future forintegrating biorefineries into industries where carbon dioxide can becaptured from point sources, for example.

A central drawback of previous methods developed to date for convertingethylene glycol to glycolic acid is the method of production is relianton a biotransformation that requires separation of the geneticallymodified microorganism and resuspension in a phosphate buffered media ordistilled water. This presents a problem for commercial applications asit is expensive to separate biomass and suspend in a fresh medium. Henceit is desirous to develop a method for producing glycolic acid in asingle fermentation vessel.

Whereas the production of glycolic acid by previous methods developed todate have relied on converting ethylene glycol in absence of nutrientsor genes that allow for cell growth, new learnings disclosed in thepresent document for producing glycolic acid is that it is not necessaryto limit cell growth either through the deactivation of the enzymeglycolate oxidase (glcDEF) or through the use of media that lacks one ormore of the following: a carbon source for growth, a nitrogen source forgrowth, a phosphate source for growth, a sulfur source for growth, tracemetals or vitamins required for growth. Furthermore, considering thatboth oxygen is necessary for growth and for glycolate production, it hasnot been shown in the past whether the presence of even small quantitiesof oxygen would allow for the production of glycolic acid since carboncould be diverted towards biomass. For this reason, the literature doesnot indicate that glycolic acid can be produced at yields greater than80% by weight in a micro-aerobic environment and with a functioningglycolate oxidase. Further still, it is disclosed that when the oxygenuptake rate of the cell is less than 6 mmol/gDW/h the fermentation mediais able to accumulate at least 80% by weight glycolic acid relative tothe ethylene glycol consumed. In addition, whereas previously disclosedmethods of glycolic acid production using a strain of Escherichia coliused a wild-type lactaldehyde reductase, we use an oxygen tolerantenzyme whose activity has been shown to be inhibitory in the presence ofoxygen and the use of an oxygen tolerant alcohol reductase is a novelembodiment of a glycolic acid producing microorganism.

It is also noted that various alternative methods can be used adaptedfrom the information described herein. For example, substrates otherthan ethylene glycol can in some cases be used as a carbon source,particularly those that are similar to ethylene glycol such as otherdiols or polyols where corresponding metabolic pathways are leveraged;microorganisms other than E. Coli can be used and can be geneticallyengineered in analogous ways as described herein, and the processes touse such microorganisms can be adapted in terms of optimizing operatingconditions such as pH, temperature, and so on; other geneticmodifications can be made in addition to those described herein; andother process operating conditions can be used depending on variousfactors (e.g., a threshold value for oxygen uptake rate other than 6mmol/gDW/h can be used to define two process phases of growth andproduction; and/or a ratio of consumption of the substrate (e.g.,ethylene glycol) in the two phases in terms of cell growth versusglycolate production; and/or other properties regarding the two phasesand the possibility of additional phases prior to or after the twophases of growth and production). In addition, some aspects of theprocesses described herein can be used to produce other products, suchas those similar to glycolate, or a mixture of products that may includeglycolate, depending on various factors.

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The references mentioned in the present document are hereby incorporatedherein by reference.

1-124. (canceled)
 125. A microorganism engineered to overexpress,relative to a corresponding wild-type microorganism, a polynucleotideencoding a zinc-dependent alcohol dehydrogenase and a polynucleotideencoding a lactaldehyde dehydrogenase, wherein overexpression of thealcohol dehydrogenase and the lactaldehyde dehydrogenase confer on themicroorganism an increased ability to produce glycolate from ethyleneglycol, relative to a corresponding microorganism that overexpressesnone or only one of the alcohol dehydrogenase and the lactaldehydedehydrogenase.
 126. The microorganism of claim 125, wherein at least oneof the alcohol dehydrogenase and the lactaldehyde dehydrogenase isheterologous with respect to the microorganism.
 127. The microorganismof claim 125, wherein both the alcohol dehydrogenase and thelactaldehyde dehydrogenase are heterologous with respect to themicroorganism.
 128. The microorganism of claim 125, wherein the alcoholdehydrogenase and the lactaldehyde dehydrogenase are expressed fromexogenous polynucleotides introduced into the microorganism.
 129. Themicroorganism of claim 128, wherein at least one of the exogenouspolynucleotides is integrated into the genome of the microorganism. 130.The microorganism of claim 125, wherein the overexpression of at leastone of the alcohol dehydrogenase and the lactaldehyde dehydrogenase isunder control of an exogenous or heterologous regulatory elementenabling control of expression in response to oxygen levels, pH,nutrient concentration, or the presence of an inducer.
 131. Themicroorganism of claim 125, wherein the overexpression of both of thealcohol dehydrogenase and the lactaldehyde dehydrogenase is undercontrol of an exogenous or heterologous regulatory element enablingcontrol of expression in response to oxygen levels, pH, nutrientconcentration, or the presence of an inducer.
 132. The microorganism ofclaim 125, wherein the microorganism expresses a functional glycolateoxidase.
 133. The microorganism of claim 125, wherein the zinc-dependentalcohol dehydrogenase is a variant of a Gluconobacter oxydans alcoholdehydrogenase, wherein the Gluconobacter oxydans alcohol dehydrogenaseis encoded by the Gluconobacter oxydans 621H GOX0313 gene.
 134. Themicroorganism of claim 125, wherein the lactaldehyde dehydrogenase is avariant of an Escherichia coli lactaldehyde dehydrogenase, wherein theEscherichia coli lactaldehyde dehydrogenase is encoded by theEscherichia coli aldA gene.
 135. The microorganism of claim 125, whichis comprised in a fermentation broth comprising ethylene glycol as acarbon source for the microorganism.
 136. The microorganism of claim125, which is comprised in a fermentation broth comprising glycolate asa fermentative product.
 137. The microorganism of claim 136, wherein theconcentration of glycolate in the fermentation broth is greater than 1g/L.
 138. The microorganism of claim 136, wherein the concentration ofglycolate in the fermentation broth is greater than 5 g/L.
 139. Themicroorganism of claim 136 wherein the concentration of glycolate in thefermentation broth is greater than 10 g/L.
 140. A microorganismcomprising a first polynucleotide encoding: a variant of Gluconobacteroxydans alcohol dehydrogenase, wherein the Gluconobacter oxydans alcoholdehydrogenase is encoded by the Gluconobacter oxydans 621H GOX0313 gene;and a variant of an Escherichia coli lactaldehyde dehydrogenase, whereinthe Escherichia coli lactaldehyde dehydrogenase is encoded by theEscherichia coli aldA gene.
 141. The microorganism of claim 140, whereinthe overexpression of at least one of the alcohol dehydrogenase and thelactaldehyde dehydrogenase is under control of an exogenous orheterologous regulatory element enabling control of expression inresponse to oxygen levels, pH, nutrient concentration, or the presenceof an inducer.
 142. The microorganism of claim 125, wherein themicroorganism expresses a functional glycolate oxidase.
 143. A methodfor improving a microorganism's tolerance to oxygen in the presence ofethylene glycol as a carbon source, the method comprisingoverexpressing, relative to a corresponding wild-type microorganism, apolynucleotide encoding an alcohol dehydrogenase that utilizes zinc as acofactor; and a polynucleotide encoding a lactaldehyde dehydrogenase,wherein overexpression of the alcohol dehydrogenase and the lactaldehydedehydrogenase confer on the microorganism an increased ability toconvert ethylene glycol to glycolate, relative to a correspondingmicroorganism that overexpresses none or only one of the alcoholdehydrogenase and the lactaldehyde dehydrogenase.
 144. The method ofclaim 143, wherein the microorganism expresses a functional glycolateoxidase.